To understand the regulation and mechanism of smooth muscle contraction and how actin and myosin interact in nonmuscle cells, we have been using various assays of myosin function. One of these, the in vitro motility assay, involves the visualization, in the fluorescent microscope, of the movement of fluorescently-labeled actin filaments over a surface coated with myosin molecules. This movement is an active process which requires the presence of myosin and MgATP. The movement of actin filaments by smooth muscle and nonmuscle myosins is almost completely dependent upon phosphorylation of the myosin by myosin light chain kinase. Calcium-dependent thin filament regulatory systems similar, but not identical, to the troponin-tropomyosin system are potentially present in smooth muscle and nonmuscle cells. One such system depends on caldesmon and calmodulin. We have shown that this system is capable of regulating the movement of actin by myosin in a calcium-dependent manner. Another system involves a more recently described smooth muscle protein, calponin, which also binds calmodulin. Calponin can also regulate the sliding of actin in vitro. The function of other actin binding proteins in smooth muscle is not understood. Filamin, an actin bundling protein, is. one such example. It exists in smooth muscle tissue in large quantities. We find that filamin is capable of exerting an internal load that is resistant to the active sliding of actin by myosin in the in vitro motility assay.